Gas Chromatography Mass Spectrometry

Gas chromatography mass spectrometry (GC/MS) is an instrumental technique,

comprising a gas chromatograph (GC) coupled to a mass spectrometer (MS), by which
complex mixtures of chemicals may be separated, identified and quantified. This makes
it ideal for the analysis of the hundreds of relatively low molecular weight compounds
found in environmental materials. In order for a compound to be analysed by GC/MS it
must be sufficiently volatile and thermally stable. In addition, functionalised compounds
may require chemical modification (derivatization), prior to analysis, to eliminate
undesirable adsorption effects that would otherwise affect the quality of the data
obtained. Samples are usually analyzed as organic solutions consequently materials of
interest (e.g. soils, sediments, tissues etc.) need to be solvent extracted and the extract
subjected to various 'wet chemical' techniques before GC/MS analysis is possible.

The sample solution is injected into the GC inlet where it is vaporized and swept onto a
chromatographic column by the carrier gas (usually helium). The sample flows through
the column and the compounds comprising the mixture of interest are separated by
virtue of their relative interaction with the coating of the column (stationary phase) and
the carrier gas (mobile phase). The latter part of the column passes through a heated
transfer line and ends at the entrance to ion source (Fig. 1) where compounds eluting
from the column are converted to ions.

Two potential methods exist for ion production. The most frequently used method is
electron ionisation (EI) and the occasionally used alternative is chemical ionization (CI).
For EI a beam of electrons ionise the sample molecules resulting in the loss of one
electron. A molecule with one electron missing is called the molecular ion and is
represented by M+. (a radical cation). When the resulting peak from this ion is seen in a
mass spectrum, it gives the molecular weight of the compound. Due to the large amount
of energy imparted to the molecular ion it usually fragments producing further smaller
ions with characteristic relative abundances that provide a 'fingerprint' for that molecular
structure. This information may be then used to identify compounds of interest and help
elucidate the structure of unknown components of mixtures. CI begins with the ionization
of methane (or another suitable gas), creating a radical which in turn will ionise the
sample molecule to produce [M+H]+ molecular ions. CI is a less energetic way of ionising
a molecule hence less fragmentation occurs with CI than with EI, hence CI yields less
information about the detailed structure of the molecule, but does yield the molecular
ion; sometimes the molecular ion cannot be detected using EI, hence the two methods
complement one another. Once ionized a small positive is used to repel the ions out of
the ionization chamber.

The next component is a mass analyser (filter), which separates the positively charged
ions according to various mass related properties depending upon the analyser used.
Several types of analyser exist: quadrupoles (Fig. 2), ion traps, magnetic sector, time-of-
flight, radio frequency, cyclotron resonance and focusing to name a few. The most
common are quadrupoles and ion traps. After the ions are separated they enter a
detector the output from which is amplified to boost the signal. The detector sends
information to a computer that records all of the data produced, converts the electrical
impulses into visual displays and hard copy displays. In addtion, the computer also
controls the operation of the mass spectrometer.

Figure 1  A schematic of an ion source

Figure 2  A schematic of a quadrupole analyser

Diagrams by Dr Paul Gates, School of Chemistry, University of Bristol

 GC/MS Analysis

 By Frederic Douglas 

patentattorney@law.com 
http://sites.netscape.net/dougfrm
  
 

Introduction

Gas chromatography ("GC") and mass spectrometry ("MS") make an effective

combination for chemical analysis.  This article serves to demonstrate tools for an
effective attack or defense of GC/MS evidence.  To effectively use GC/MS evidence one
must understand the process.  First, the GC process will be considered, then the MS
instrument will be presented.  After a background in GC and MS is obtained, the reader
will learn how to analyze the evidence produced by these instruments.  The focus of this
article lies in presenting the limitations to GC/MS analysis.

Gas Chromatography

GC analysis is a common confirmation test.   Among its uses are drug testing and
environmental contaminant identification.  GC analysis separates all of the components
in a sample and provides a representative spectral output.   The technician injects the
sample into the injection port of the GC device.  The GC instrument vaporizes the
sample and then separates and analyzes the various components.   Each component
ideally produces a specific spectral peak that may be recorded on a paper chart or
electronically.   The time elapsed between injection and elution is called the "retention
time."  The retention time can help to differentiate between some compounds.  The size
of the peaks is proportional to the quantity of the corresponding substances in the
specimen analyzed.  The peak is measured from the baseline to the tip of the peak.

Imagine a pile of different types of balls resting at the bottom of an inclined, paved
driveway.  This pile includes ball bearings, marbles, ping pong balls, golf balls, wiffle
balls, handballs, tennis balls, hockey pucks, baseballs, soccer balls, volley balls,
basketballs, footballs, and bowling balls.  Attempt to move this motley collection of balls
up the driveway with a normal leafblower.  Some of the pile will quickly move to the top
of the driveway immediately, some balls will migrate at varying speeds, and some balls
may take an eternity to reach the end of the driveway.

The difference in the time that each type of ball takes to travel to the top depends upon
the characteristics of each ball.  Obviously, the lighter balls travel more quickly.  Also,
some balls may take longer due to their shape, like the hockey puck or the football.  The
different balls interact with each other as the air from the leaf blower acts on the pile. 
This interaction may hinder or accelerate the ball's travel as the balls strike each other. 
The surface characteristics of the ball may be important, as in the examples of the tennis
ball and golf ball.

GC analysis depends on similar phenomena to separate chemical substances.  A

mixture of chemicals present in a specimen can be separated in the GC column.  Some
chemical and physical characteristics of the molecules cause them to travel through the
column at different speeds.  If the molecule has low mass it may travel more swiftly. 
Also, the molecule's shape may affect the time needed to exit the column.  How the
different substances relate to each other may cause the time needed to travel the
column to increase or decrease.  Interactions between the sample's molecule and the
column surface may cause the molecule to be retained inside the column for a different
amount of time than similar molecules that interact with the column differently.

Description of Process

The equipment used for gas chromatography generally consists of an injection port at
one end of a metal column packed with substrate material and a detector at the other
end of the column.  A carrier gas propels the sample down the column.   The technician
uses flow meters and pressure gauges to maintain a constant gas flow.   A gas that
does not react with the sample or column is essential for reliable results.   For this
reason, carrier gases are usually argon, helium, hydrogen, nitrogen, or hydrogen.  Many
analysts use helium because it does not react.   Hydrogen usually is a good carrier gas
but it may react and convert the sample into another substance.   The ultimate choice for
a carrier gas may depend on the type of detector used.

To ensure proper separation, the sample must enter  the column in a discreet, compact
packet.   Normally the sample is injected into the injection port with a hypodermic needle
and syringe capable of measuring the specimen amount.   The needle is stuck into a
replaceable neoprene or silicone rubber septum that covers the injection port.   The
injection port is maintained at a temperature at which the sample vaporizes
immediately.   Ideally, the sample spreads evenly along the cross section of the column,
forming a plug.

The column is a metal tube, often packed with a sand-like material to promote maximum
separation.   Columns are commonly obtained pre-packed by vendors.   As the sample
moves through the column, the different molecular characteristics determine how each
substance in the sample interacts with the column surface and packing.  The column
allows the various substances to partition themselves.

Substances that do not like to stick to the column or packing move through the column
rapidly.   Substances that do not like to stick to the column or packing are impeded but
eventually elute from the column.   Ideally, the various components in the sample
separate before eluting from the column end.

The GC instrument uses a detector to measure the different compounds as they emerge
from the column.   Among the available detectors are the argon ionization detector,
flame ionization detector, flame emission detector, cross section detector, thermal
conductivity detector, and the electron capture detector.   Choosing the proper detector
depends upon the use.   Some considerations are that the flame detectors destroy the
sample, the thermal conductivity detector is universally sensitive, and the argon
ionization detector requires argon as a carrier gas.   The spectral output is usually stored
electronically and displayed on a monitor.  The technician can produce a hard copy
record.

The argon ionization detector does not detect water, carbon tetrachloride, nitrogen,
oxygen, carbon dioxide, carbon monoxide, ethane, or compounds containing fluorine.  
The flame ionization detector does not respond to water, nitrogen, oxygen, carbon
dioxide, carbon monoxide, helium, or argon.    If a specimen contains water, a flame
ionization detector should be used.   The electron capture detector can not detect simple
hydrocarbons but does detect compounds containing halides, nitrogen, or phosphorus.

Retention Time

The amount of time that a compound is retained in the GC column is known as the
retention time.  The technician should measure retention time from the sample injection
until the compound elutes from the column.  The retention time can aid in differentiating
between some compounds.  However, retention time is not a reliable factor to determine
the identity of a compound.   If two samples do not have equal retention times, those
samples are not the same substance.   However, identical retention times for two
samples only indicate a possibility that the samples are the same substance.  
Potentially thousands of chemicals may have the same retention time, peak shape, and
detector response.   For example, under certain conditions, DDT has the same retention
time as PCBs (polychlorinated biphenyls).   Some believe that environmental testing
showed erroneously high amounts of DDT.   GC instruments showed only one peak for
what is believed to be a mixture of DDT and PCBs.   This experimental data led to the
banning of DDT in the U.S.    Bluntly, GC is "one of the quickest ways of getting the
wrong answer in qualitative analysis."

Quality Assurance/Quality Control Procedures

Before analyzing a sample, the technician should tune and calibrate the instrument.  
Tuning can be accomplished using specific concentrations of
Decafluorotriphenylphosphine and p-Bromofluorobenzene.  A technician can process a
spiked sample (containing a known concentration of a substance) to check calibration
and tuning.   If the GC/MS instrument does not detect the substance or shows a greater
or lesser concentration than the known concentration, the technician must recalibrate the
instrument.   Also, the technician can use a blank sample (containing no detectable
compounds) to test the GC/MS instrument's data reporting accuracy.   If the device
indicates the presence of a substance in the blank sample, the device may contain
residue from prior analysis.   If this occurs, the technician must retune and recalibrate
the GC/MS instrument.

    Proper scientific practice requires that the GC technician compare the spectral output
with a known standard sample of the suspected substance.   The standard sample must
be analyzed with the same instrument, under the same conditions, immediately before
and immediately after analyzing the unknown specimen.   If the resulting three spectral
outputs do not agree, the technician can not make a reliable identification of the
specimen based on the GC analysis.

Analysis of Output

    Less than ideal spectral peaks may indicate less than ideal analytical procedures or
equipment.  The technician can readily observe whether the output exhibits
unsatisfactory results.  Ideally, the spectral peaks should be symmetrical, narrow,
separate (not overlapping), and made with smooth lines.  GC evidence may be suspect
if the peaks are broad, overlapping, or unevenly formed.  If a poorly shaped peak
contains a steep front and a long, drawn-out tail, this may indicate traces of water in the
specimen.

    The GC technician should inject the specimen into the septum rapidly and smoothly to
attain good separation of the components in a specimen.  If the technician injects the
specimen too slowly, the peak may be broad or overlap.  A twin peak may result from
the technician hesitating during the injection.  A smoothly performed injection, without
abrupt changes, should result in a smoothly formed peak.  A twin peak may also indicate
that the technician injected two specimens consecutively. 
 

Limitations

 Response Factor

    The size of a spectral peak is proportional to the amount of the substance that
reaches the detector in the GC instrument.   No detector responds equally to different
compounds.   Results using one detector will probably differ from results obtained using
another detector.  Therefore, comparing analytical results to tabulated experimental data
using a different detector does not provide a reliable identification of the specimen.

    A "response factor" must be calculated for each substance with a particular detector.  
A response factor is obtained experimentally by analyzing a known quantity of the
substance into the GC instrument and measuring the area of the relevant peak.   The
experimental conditions (temperature, pressure, carrier gas flow rate) must be identical
to those used to analyze the specimen.   The response factor equals the area of the
spectral peak divided by the weight or volume of the substance injected.   If the
technician applies the proper technique, of running a standard sample before and after
running the specimen, determining a response factor is not necessary.

 Worn Septum

    An injection port septum should last between 100 and 200 injections.   Higher
injection port temperatures shorten the septum's lifespan.   A leaking septum adversely
affects the GC instrument's sensitivity.

    If a portion of the specimen leaks back out of the septum, the amount of the specimen
is not recorded.  This event makes any eventual quantitative result erroneous.  If air
should leak into the injection port through a worn septum, the oxygen and water
contained in air may skew the results.  Any oxygen may react with the specimen
components.  If this happens, the GC instrument will provide results indicating the
presence of this unintended reaction product, instead of the original compounds present
in the specimen vial.  Any water in the column adversely affects the GC instrument's
ability to separate components.

 Injection Port Temperature

    The temperature of the GC injection port must be high enough to vaporize a liquid
specimen instantaneously.   If the temperature is too low, separation is poor and broad
spectral peaks should result or no peak develops at all.   If the injection temperature is
too high, the specimen may decompose or change its structure.  If this occurs, the GC
results will indicate the presence of compounds that were not in the original specimen.

 Residual Impurities

    Ideally, all components of a specimen elute completely from the GC column.  If any
substance remains inside the column, the substance may elute during subsequent
analyses with other specimens.  This may result in an unexpected peak in the output. 
The peak produced should be broad.

 Carrier Gas

    If the GC instrument uses hydrogen for the carrier gas, the technician must consider
whether the hydrogen may react with any of the compounds present in the specimen.  If
the hydrogen does react, a broad peak will result.  When using a thermal conductivity
detector, care should be taken as a false peak may occur if the carrier gas's thermal
conductivity is in the range of the thermal conductivity of any compound in the
specimen.  An unstable carrier gas flow rate may produce a drifting baseline and false
broad peaks.  A carrier gas should be pure.  Regular changing of the gas filter should
prevent significant impurities.

Crucial Factors

    GC analysis is highly reliable if the instrument is properly maintained, the technician
follows proper procedures, and the interpretation of the results is competent.  While
some factors rarely affect GC analysis, some factors are absolutely essential for the use
of reliable GC evidence.  In all cases a technician must process a standard sample
containing a verified composition identical to the presumed contents of the collected
specimen.  This standard sample must be processed before and after the collected
specimen under identical conditions.  Any output from the collected specimen that does
not match the standard sample is inconclusive.  If tabulated reference data exists for the
relevant conditions, the specimen data must match the reference data.

    If advance notice of GC testing is available, an adverse party should observe the
procedure.  If a retained consultant or the knowledgeable attorney observes the
technician's use of the GC instrument, important information can be recorded.  The
technician's preparation of the specimen and the subsequent injection can be observed
for errors or malfunctioning equipment.  The observer should record the instrument's
make, model, serial number, injection temperature, column temperature, carrier gas flow
rates and pressure, identify the type of detector used, and observe any manipulation of
the data by use of a computer.   Ensure that the technician properly starts measuring the
time at injection and records the time of elution.  Any discrepancy in the time will
produce an erroneous retention time.  If the procedure can not be observed, the adverse
party should seek all pertinent information (experimental conditions, measurements,
instrument identification) and hard copy output.

Mass Spectrometry (MS)

 MS analysis is commonly used in arson investigations, engine exhaust analysis,

petroleum product analysis, and for blood monitoring in surgery.  MS identifies
substances by electrically charging the specimen molecules, accelerating them through
a magnetic field, breaking the molecules into charged fragments and detecting the
different charges.  A spectral plot displays the mass of each fragment.  A technician can
use a compound's mass spectrum for qualitative identification.   The technician uses
these fragment masses as puzzle pieces to piece together the mass of the original
molecule, the "parent mass."

 The parent mass is analogous to the picture on top of a puzzle box, a guide to the end
result obtained by putting together the fragment masses, or puzzle pieces.  From the
molecular mass and the mass of the fragments, reference data is compared to
determine the identity of the specimen.  Each substance's mass spectrum is unique. 
Providing that the interpretation of the output correctly determines the parent mass, MS
identification is conclusive. 
 

Description of Process

    Today many different types of MS instruments exist, each one using a different
apparatus and process for producing mass spectra.   This article's description of the MS
process will limit itself to a basic description of a conventional large magnet mass
spectrometer.  Such a MS instrument contains a sample inlet, an ionization source, a
molecule accelerator, and a detector.

    MS analysis requires a pure gaseous sample.  The sample inlet is maintained at a
high temperature, up to 400° C (752° F), to ensure that the sample stays a gas.    Next
the specimen enters the ionization chamber.   A beam of electrons is accelerated with a
high voltage.   The specimen molecules are shattered into well-defined fragments upon
collision with the high voltage electrons.   Each fragment is charged and travels to the
accelerator as an individual particle. 
 In the acceleration chamber the charged particle's velocity increases due to the
influence of an accelerating voltage.   For one value of voltage only one mass
accelerates sufficiently to reach the detector.   The accelerating voltage varies to cover a
range of masses so that all fragments reach the detector.

    The charged particles travel in a curved path towards the detector.   When an
individual charged particle collides with the detector surface, several electrons (also
charged particles) emit from the detector surface.   Next, these electrons accelerate
towards a second surface, generating more electrons, which bombard another surface. 
Each electron carries a charge.   Eventually, multiple collisions with multiple surfaces
generate thousands of electrons which emit from the last surface.   The result is an
amplification of the original charge through a cascade of electrons arriving at the
collector.   At this point the instrument measures the charge and records the fragment
mass as the mass is proportional to the detected charge.

    The MS instrument produces the output by drawing a array of peaks on a chart, the
"mass spectrum."   Each peak represents a value for a fragment mass.   A peak's height
increases with the number of fragments detected with one particular mass.   As in the
case of the GC detectors, a peak may differ in height with the sensitivity of the detector
used.

Analysis of Output

    Each substance has a characteristic mass spectrum under particular controlled
conditions.   A technician can identify a specimen by comparing the specimen's mass
spectrum with known compounds.   Quantitative analysis is possible by measuring the
relative intensities of the mass spectra.

    Usually a mass spectrum will display a peak for the unfragmented molecule of the
specimen.   This is commonly the greatest mass detected, called the "parent mass."  
Like the picture on a puzzle box, the parent mass is used to fit the pieces together from
the other peaks in the mass spectrum.  The parent mass reveals the mass of the
molecule while the other peaks indicate the molecule's structure.

    Determining the parent peak and consequently the molecular mass of the specimen is
the most difficult part of MS analysis.   Identifying the parent mass is outside the scope
of this article.   Assuming that a technician can correctly determine the molecular mass,
the technician makes an educated guess of the specimen's identity and compares the
mass spectrum to reference spectra for confirmation.   The mass spectra for larger
molecules containing carbon are complicated and require tedious calculations that are
subject to error.   Computers are commonly used for spectral analysis.

Limitations

 Resolution 
 The "resolution" is a value that represents the instrument's ability to distinguish two
particles of different masses.   The greater the MS instrument's resolution, the greater its
usefulness for analysis.   An MS instrument provides more accurate results for larger
molecules when the instrument has a high resolution.   A high resolution MS instrument
is advisable for analyzing body fluids because they have high molecular masses. A low
resolution MS instrument may not sufficiently characterize a large mass substance.

 Pressure

 If the interior pressure in an MS instrument is too high, erroneous results may occur.  As
the specimen molecule breaks up, the fragments accelerate.  If a fragment collides with
another fragment, then these two fragments may combine to make a new particle.   In
this event, the detector will register the mass of this new particle on the mass spectrum.  
The reference spectra for comparison are produced under low pressure conditions which
minimize collisions between fragments.  A technician would find a spectral peak where
one is not expected.  In the puzzle analogy, this is similar to finding pieces from a
different puzzle in your box and trying to make these extraneous pieces fit.  As this is
impossible, any MS analysis under high pressure conditions would depend greatly on
guesswork by the technician.

 Parent Mass

 Finding the correct parent peak in the mass spectra may be difficult.  Finding the parent
peak helps to determine the parent mass, which should lead to determining the
specimen's molecular mass.   For high molecular mass compounds, like drugs and body
fluids, a parent peak is often not observed.  This makes qualitative identification difficult. 
A special type of MS, chemical ionization MS, reduces the likelihood of missing the
parent mass.
 High Speed Scanning

 High speed scanning MS instruments are able to rapidly analyze specimens.   However,
the increased speed is a tradeoff for decreased resolution.   Quantitative measurements
are unreliable with high speed scanning.

 Technician's Skills

 As in the puzzle analogy, knowing the shape of a piece of the molecule helps to join the
pieces together.  To determine the specimen's molecular structure before fragmentation,
the technician needs to employ skill and art to determine the molecular structure from
mass spectra patterns.   Computers and databases can assist, but a human expert is
necessary to distinguish between likely and unlikely answers.   Alone, a computer can
not determine molecular structures as well as a competent human.   This causes the
weight of MS evidence to depend greatly on the technician's qualifications and
proficiency with MS spectrum analysis.

Crucial Factors

  MS analysis is highly reliable if the instrument is of sufficient resolution and the

technician's interpretation of the results is competent.  While some factors rarely affect
MS analysis, some factors are absolutely essential for the use of reliable MS evidence. 
In all cases a technician must process a standard sample containing a verified
composition identical to the presumed contents of the collected specimen.  This
standard sample must be processed under identical conditions, both before and after
processing the collected specimen .  Any identification based on output from the
collected specimen that does not match the standard sample is inconclusive.

 Because MS is highly sensitive, care should be taken that not even the slightest trace of
a previous sample remain within the MS instrument.   The technician should run a
"background spectrum," an analysis without a specimen, before analyzing the specimen
in question.   This practice is the only way that an independent analyst can definitely
interpret MS output.

 If tabulated reference data exists for the relevant conditions, the specimen data must
match the reference data.  Despite the use of sophisticated instruments, computers, and
proficient personnel, there is always some doubt in conclusions based on interpretation
of mass spectra.   One example is the pair of narcotic compounds, N-methyl-3-
piperidylbenzilate and N-methyl-4-piperidylbenzilate.   The compounds have the same
molecular mass but differ in the position of one molecular group.   In some instances,
two molecules that only differ in structure may be separated with the proper instrument
and technique.

    If advance notice of MS testing is available, an adverse party should observe the
procedure.  If a retained consultant or the knowledgeable attorney observes the
technician's use of the MS instrument, important information can be recorded.  The
observer should record the instrument's make, model, serial number, resolution,
pressure, and identify the type of detector used.

    It is important to observe and record which possible compounds the computerized
database produced.  As the technician uses personal judgment to rule out these other
compounds, an adverse attorney should consider the likelihood that one of the other
contending identifications may be the proper choice.  If the procedure can not be
observed, the adverse party should seek all pertinent information (experimental
conditions, measurements, instrument identification) and hard copy output.

    In all instances, hard copy data is essential.  The mass spectra should include the
scale of mass units reported.   This enables an independent analyst to check whether
the specimen in question contains major molecular fragments reported in the literature.  
The mass spectra should also include the MS pressure and the accelerating voltage.  
An independent analyst can use the operating conditions to resolve whether
discrepancies in the mass spectrum arise from misidentification or from instrumental
malfunction.   Providing a properly labeled printing of the mass spectra is easy, not time-
consuming, and of minimal cost.

GC/MS Combination

 The GC device is generally a reliable analytical instrument.  The GC instrument is

effective in separating compounds into their various components. However, the GC
instrument can not be used for reliable identification of specific substances.  The MS
instrument provides specific results but produces uncertain qualitative results.  When an
analyst uses the GC instrument to separate compounds before analysis with an MS
instrument, a complementary relationship exists.  The technician has access to both the
retention times and mass spectral data.  Many scientists consider GC/MS analysis as a
tool for conclusive proof of identity.

    GC/MS analysis, where the effluent to the GC instrument is the feed to the MS
instrument, is in wide use for confirmation testing of substances.  Drug testing,
manufacturing quality control and environmental testing are some typical uses.

Limitations

 Although many consider GC/MS to be the "gold standard" in scientific analysis, GC/MS
does have some limitations.  Because great faith is maintained in GC/MS analysis,
erroneous results are not expected and hard to dispute.  However, false positives and
false negatives are possible.

    Some problems with GC/MS originate in improper conditions in the GC portion of the
analysis.  If the GC instrument does not separate the specimen's compounds
completely, the MS feed is impure.  This usually results in background "noise" in the
mass spectrum.  If the carrier gas in the GC process is not correctly deflected from
entering the MS instrument, similar  contamination may occur.

    Also, the MS portion suffers from the inexact practice of interpreting mass spectra.  
An analyst must correlate computer calculations with system conditions.  The typical
memory bank for MS identification contains about 5000 spectra for a particular group of
compounds.   Even if a competent analyst could find conclusive results pointing to one
substance out of 5000 substances, this does not rule out the remaining over 200,000
known existing chemicals.   For the 5000-spectra memory bank, the typical computer
result is limited to as many as six possible identifications.

    In one instance, erroneous GC/MS results may have been responsible for a criminal
defendant receiving a death sentence.  John Brown killed a police officer and wounded
two bar patrons in a shoot-out on June 7, 1980 in Garden Grove, California.   Mr.
Brown's diminished capacity defense to capital murder relied on the assertion that Mr.
Brown was under the influence of narcotics at the time of the shooting.    The prosecution
introduced GC/MS evidence that showed Mr. Brown's blood to be free of narcotics.   The
California Supreme Court overturned the jury's death sentence because the prosecution
never introduced evidence from a radioactive immunoassay ("RIA") test that detected
phencyclidine (PCP) in Mr. Brown's blood.   Obviously, an example like this
demonstrates that analytical evidence, including GC/MS, should always be confirmed
with another reliable technique.

    A more advanced analytical method is MS/MS, a tandem series of instruments, which
has the advantage of increased sensitivity.   One court states that MS/MS analysis has
never produced a false positive in the FBI laboratory.   However, MS/MS is not widely
used yet as the instrument's cost is prohibitive.

Conclusion

    GC and MS are useful tools for chemical analysis, especially when used together.   An
attorney can present an effective attack or defense of GC/MS evidence with a basic
knowledge of the analysis process and an insistence on documentation of important
indicators that may affect GC/MS results.  At the minimum, a technician must process
standard samples before and after analyzing a specimen in question.  In litigation an
adverse party should seek hard copy output, including system conditions.  Finally, no
analytical technique produces results that are completely without doubt.  An effective
advocate should always seek corroboration of GC/MS results. 
 
SRIF's GC-MS System

Gas Chromatography-Mass Spectroscopy

Background

Note: This GC-MS background is from last year's bioinstrumentation class.

Introduction

Gas chromatography-mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical

techniques. As the name implies, it is actually two techniques that are combined to form a single
method of analyzing mixtures of chemicals. Gas chromatography separates the components of a
mixture and mass spectroscopy characterizes each of the components individually. By combining
the two techniques, an analytical chemist can both qualitatively and quantitatively evaluate a
solution containing a number of chemicals.
The uses for GC-MS are numerous. They are used extensively in the medical, pharmacological,
environmental, and law enforcement fields. This laboratory exercise will look at how
environmental chemists evaluate samples containing the pollutants called PAHs.

Gas Chromatography

In general, chromatography is used to separate mixtures of chemicals into individual components.

Once isolated, the components can be evaluated individually.

In all chromatography, separation occurs when the sample mixture is introduced (injected) into
a mobile phase. In liquid chromatography (LC), the mobile phase is a solvent. In gas
chromatography (GC), the mobile phase is an inert gas such as helium.

The mobile phase carries the sample mixture through what is referred to as a stationary phase.
The stationary phase is a usually chemical that can selectively attract components in a sample
mixture. The stationary phase is usually contained in a tube of some sort. This tube is referred to
as a column. Columns can be glass or stainless steel of various dimensions.

The mixture of compounds in the mobile phase interacts with the stationary phase. Each
compound in the mixture interacts at a different rate. Those that interact the fastest will exit (elute
from) the column first. Those that interact slowest will exit the column last. By changing
characteristics of the mobile phase and the stationary phase, different mixtures of chemicals can
be separated. Further refinements to this separation process can be made by changing the
temperature of the stationary phase or the pressure of the mobile phase.

Our GC has a long, thin column containing a thin interior coating of a solid stationary phase (5%
phenyl-, 95% dimethylsiloxane polymer). This 0.25 mm diameter column is referred to as a
capillary column. This particular column is used for semivolatile, non-polar organic compounds
such as the PAHs we will look at. The compounds must me in an organic solvent.

The capillary column is held in an oven that can be programmed to increase the temperature
gradually (or in GC terms, ramped). this helps our separation. As the temperature increases, those
compounds that have low boiling points elute from the column sooner than those that have higher
boiling points. Therefore, there are actually two distinct separating forces, temperature and
stationary phase interactions mentioned previously.

As the compounds are separated, they elute from the column and enter a detector. The detector is
capable of creating an electronic signal whenever the presence of a compound is detected. The
greater the concentration in the sample, the bigger the signal. The signal is then processed by a
computer. The time from when the injection is made (time zero) to when elution occurs is
referred to as the retention time (RT).

While the instrument runs, the computer generates a graph from the signal. (See figure 1). This
graph is called a chromatogram. Each of the peaks in the chromatogram represents the signal
created when a compound elutes from the GC column into the detector. The x-axis shows the RT,
and the y-axis shows the intensity (abundence) of the signal. In Figure 1, there are several peaks
labeled with their RTs. Each peak represents an individual compound that was separated from a
sample mixture. The peak at 4.97 minutes is from dodecane, the peak at 6.36 minutes is from
biphenyl, the peak at 7.64 minutes is from chlorobiphenyl, and the peak at 9.41 minutes is from
hexadecanoic acid methyl ester.

Figure 1: Chromatogram generated by a GC.

If the GC conditions (oven temperature ramp, column type, etc.) are the same, a given compound
will always exit (elute) from the column at nearly the same RT. By knowing the RT for a given
compound, we can make some assumptions about the identity of the compound. However,
compounds that have similar properties often have the same retention times. Therefore, more
information is usually required before an analytic al chemist can make an identification of a
compound in a sample containing unknown components.

Mass Spectroscopy

As the individual compounds elute from the GC column, they enter the electron ionization (mass
spec) detector. There, they are bombarded with a stream of electrons causing them to break apart
into fragments. These fragments can be large or small pieces of the original molecules.

The fragments are actually charged ions with a certain mass. The mass of the fragment divided by
the charge is called the mass to charge ratio (M/Z). Since most fragments have a charge of +1, the
M/Z usually represents the molecular weight of the fragment.

A group of 4 electromagnets (called a quadrapole, focuses each of the fragments through a slit
and into the detector. The quadropoles are programmed by the computer to direct only certain
M/Z fragments through the slit. The rest bounce away. The computer has the quadrapoles cycle
through different M/Z's one at a time until a range of M/Z's are covered. This occurs many times
per second. Each cycle of ranges is referred to as a scan.

The computer records a graph for each scan. The x-axis represents the M/Z ratios. The y-axis
represents the signal intensity (abundance) for each of the fragments detected during the scan.
This graph is referred to as amass spectrum (see Figure 2).

Figure 2: Mass-spectrum generated by an MS.

The mass spectrum produced by a given chemical compound is essentially the same every time.
Therefore, the mass spectrum is essentially a fingerprint for the molecule. This fingerprint can be
used to identify the compound. The mass spectrum in Figure 2 was produced by dodecane. The
computer on our GC-MS has a library of spectra that can be used to identify an unknown
chemical in the sample mixture. The library compares the mass spectrum from a sample
component and compares it to mass spectra in the library. It reports a list of likely identifications
along with the statistical probability of the match.

GC-MS

When GC is combined with MS, a powerful analytical tool is created. A researcher can take an
organic solution, inject it into the instrument, separate the individual components, and identify
each of them. Furthermore, the researcher can determine the quantities (concentrations) of each of
the components.

Figure 3 represents a three-dimensional graph generated when the GC is combined with the MS.
Try to visualize how the chromatogram combines with the mass spectrum to produce this image.
It is important for you to be able to picture this 3D image and translate it into the previous 2D
graphs. (This image is not made from the same compounds in the previous figures.) Note that you
can create either a mass spectrum or a chromatogram by making the appropriate cross section of
this 3D image. Try to visualize which cross section would produce a spectrum and which would
produce a chromatogram.

Figure 3: 3D Depiction of GC-MS output.

GAS CHROMATOGRAPHY / MASS SPECTROSCOPY

DESCRIPTION OF TECHNIQUE

Total Ion Chromatogram for Two Vegetable Oils

Gas chromatography / mass spectrometry (GC/MS) is the marriage of two analytical methods into a versatile
technique for the identification of complex volatile materials. Gas chromatography (GC) effectively separates the
different constituents of the sample for subsequent analysis and identification by mass spectrometry (MS).

The chromatographic separation relies on the interaction of the sample with a mobile phase and a stationary phase
within the GC instrument column. The sample is carried through the column by the mobile phase, typically an inert
gas. However, the sample is slowed in its travel through the column as the sample molecules repeatedly adsorb and
desorb from the stationary phase in the column. The affinity of a particular molecule for the stationary phase
determines the retention time of that constituent in the column. The molecules for each component of the sample will
travel through the column at nearly the same rate and exit (elute) from the column within a narrow time band that is
specific to that component.
Thus, compounds with different retention times in the column are physically separated for presentation to a detector
and analyzer.

The typical GC capillary column consists of a smalldiameter tube with a thin film of a high-molecularweight polymer
coated on the inside. The polymer is the stationary phase for the chromatographic process. The mobile phase can be
any inert gas, but is typically helium. The instrument also includes a heated injection port to vaporize all volatile
constituents of the sample and an oven to keep the constituents in gas form as they pass through the column.

As a sample constituent elutes from the GC column, it enters the ionization chamber of the mass spectrometer where
the molecules are ionized, typically by electron impact. When an electron impact with a sample molecule results in the
loss of an electron from the molecule, a positive ion is formed. Some of the molecular ions are further fragmented
into daughter ions and neutral fragments.

The positive ions are then repelled out of the ionization chamber by a small positive charge within the chamber.
Negative ions are also formed by the electron impact, but are not analyzed. The positive ions are separated according
to their mass by a mass analyzer. The mass analyzer most commonly used in GC/MS is the quadrapole filter, in which
the ions pass by four hyperbolic magnetic poles created by a radio frequency field. The magnetic poles separate the
ions by their mass/charge ratio, successively focusing ions with increasing mass onto a detector for counting. The
analyzer scans step-wise through a set range of mass values to evaluate the relative abundance of ions at each mass
value. The quadrapole filter can perform a complete mass scan within the duration of a single GC elution band.

ANALYTICAL INFORMATION
 Chromotagram for Outgassing Compounds from Two Epoxies

Material Identification - The first result from the compiled data is a total-ion chromatogram (TIC), which is a plot
of the total mass eluting from the GC and detected by MS as a function of time. Each peak or band in the
chromatogram represents a discrete chemical compound, or a mixture of compounds with identical retention times.
The retention times in the chromatogram provide the first indication of the sample constituents.

More specific identification of the compound(s) for each band can then be made from the mass spectrum
corresponding to the band. Compounds are identified from the mass spectrum by their unique ion fragmentation
patterns. This compound identification analysis is performed by a computerized comparison of the mass spectra for
the sample with spectra library for known
compounds.

Quantitation - The analysis results can be quantified using the data from the chromatogram. The area under each
peak in the chromatogram is proportional to the concentration of the ompounds represented by that peak. The
concentration for each compound in the sample is calculated from a standard curve of known concentrations
established for that compound. The analysis sensitivity can be as low as a few nanograms.

TYPICAL APPLICATIONS

• Identification of foreign material contamination

• Analysis of outgassing products for disk drive components
• Identification of polymer additives
• Analysis of polymer cure by-products

SAMPLE REQUIREMENTS
The samples for GC/MS can be gases, liquids, or solids. However, only those constituents that are gaseous and stable
at the analysis temperature can be analyzed. Gases and liquids can be injected directly into the sample injector.
Volatile compounds from solid materials are collected using a headspace sampler. The sample from a headspace
attachment can be collected and injected at one time (static headspace), or the sample can be collected and analyzed
continuously over a set time period (dynamic headspace).